Genetic deletion of nitric oxide synthase 2 ameliorates Parkinson’s disease pathology and neuroinflammation in a transgenic mouse model of synucleinopathy

Studies of mouse models of Alzheimer's disease (AD) have demonstrated that nitric oxide synthase 2 (NOS2) is involved in AD pathology. However, the effects of NOS2 on the pathology of Parkinson’s disease (PD) are not well studied. To address this gap, we examined the impact of NOS2 on disease-associated phenotypes in a mouse model of PD. Transgenic mice carrying the A53T mutation of α-synuclein (SynA53T) and newly generated double transgenic mice with deletion of NOS2 (SynA53T/NOS2−/−) were used. Compared with SynA53T mice, the loss of nos2 decreased α-synuclein phosphorylation at serine 129 and reduced α-synuclein-induced microglial and astrocyte activation in SynA53T/NOS−/− mice. Additionally, neuroinflammation-related gene clusters in the deep mesencephalic nucleus (DpMe) were altered in SynA53T/NOS−/− mice compared with SynA53T mice. Taken together, our results suggest that deletion of nos2 alleviates α-synuclein pathology and α-synuclein-associated neuroinflammatory responses in the brain. Supplementary Information The online version contains supplementary material available at 10.1186/s13041-023-00996-1.


Main text
Nitric oxide (NO) is a bioactive free radical that is involved in various physiological and pathological processes in several organ systems and the central nervous system (CNS) [1]. In the brain, nitric oxide synthase 2 (NOS2) plays an important role in neurotransmission, neural development, and the immune defense response [2]. Interestingly, several recent studies have reported that NOS2 differentially regulates Alzheimer's disease (AD) pathology. For instance, deletion of nos2 in mice results in the expression of mutant amyloid precursor protein (APP) and hyperphosphorylation of tau in the brain [3]. Compared with APPSwDI mice, APPSwDI/ NOS2 −/− mice exhibit spatial memory impairment and tau pathology [4]. However, the effects of NOS2 on α-synuclein-induced Parkinson's disease (PD) pathology remain unclear.
Since genetic deletion of nos2 diminished α-synuclein aggregation in the brain, we further investigated the impact of nos2 deletion on α-synuclein-induced glial activation. The brains of 10-to 11-month-old nTg, Syn A53T , and Syn A53T /NOS2 −/− mice were subjected to immunofluorescence staining with anti-Iba-1 and anti-GFAP antibodies. Compared with nTg mice, microglial/astrocyte fluorescence intensity, the number of Iba-1-positive cells, and the Iba-1/GFAP % area were increased in the DpMe and Gi but not in the SN in Syn A53T mice (Fig. 1C, D and Additional file 1: Fig. S3). Importantly, Iba-1 fluorescence intensity, the number of Iba-1-positive cells, and the Iba-1-positive % area in the SN, DpMe, and Gi were significantly lower in Syn A53T /NOS2 −/− mice than in Syn A53T mice (Fig. 1C, D and Additional file 1: Fig.  S3). Moreover, GFAP fluorescence intensity in the SN, DpMe, and Gi was significantly reduced in Syn A53T / NOS2 −/− mice compared with Syn A53T mice ( Fig. 1E, F). The α-synuclein-induced number of GFAP-positive cells and GFAP-positive % area in the DpMe and Gi were significantly diminished in Syn A53T /NOS2 −/− mice compared with Syn A53T mice (Additional file 1: Fig. S3). In addition, Iba-1/GFAP fluorescence intensity, the number of Iba-1/GFAP-positive cells and the Iba-1/GFAP % area in the cortex, CPu, and hippocampus were significantly reduced in Syn A53T /NOS2 −/− mice compared with nTg and Syn A53T mice (Figs. S4-S5). Taken together, these data suggest that deletion of nos2 diminishes α-synucleinstimulated microglial and astrocyte activation and that NOS2 is required for α-synuclein-mediated neuroinflammation in the brain.
To systematically investigate the cellular processes affected by nos2 deletion, we classified the 1,339 DEGs into 6 clusters (C1-6) based on their differential expression in the two comparisons (Fig. 1H). C2 was upregulated in Syn A53T mice compared with nTg mice but downregulated in Syn A53T /NOS2 −/− mice compared with Syn A53T mice. Thus, we focused on this cluster because it likely includes genes associated with the effects of NOS2 on PD pathology. The cellular processes represented by the DEGs in C2 were identified by gene set enrichment analysis using Consensus Path DB [5]. Interestingly, the DEGs in C2 were mainly involved in neuroinflammatory responses, glial cell proliferation, oxidative stress, and apoptosis (Fig. 1I). Notably, genes involved in neuroinflammatory response-related processes were strongly downregulated in Syn A53T /NOS2 −/− mice compared with Syn A53T mice (Fig. 1J).
In summary, α-synuclein phosphorylation, α-synucleininduced neuroinflammation, and the expression of related genes were significantly suppressed in the brains of Syn A53T /NOS2 −/− mice. Overall, our results suggest that NOS2 is a crucial regulator of the synucleinopathy and neuroinflammatory response associated with PD pathology.
A recent study demonstrated that NOS2 overexpression induces NO production and α-synuclein aggregation in PC12 neurons [6]. In SH-SY5Y cells, NOS2 expression induces the formation of cytotoxic nitrated α-synuclein [7]. However, the effects of nos2 deletion on α-synuclein pathology have not been investigated. The significant reduction in p-Syn ser129 levels in Syn A53T /NOS2 −/− mice compared with Syn A53T mice suggests that decreasing NOS2 expression may help alleviate α-synucleinopathy in the brain.
Interestingly, several recent studies have shown that NOS2 regulates neuroinflammatory responses in the brain. For instance, the lipopolysaccharide (LPS)-induced increase in TNF-α levels is significantly reduced in nos2 knockout mice [8], and deletion of nos2 decreases the number of Iba-1/GFAP-positive cells in the brain compared with wild-type mice [9]. In addition, GFAP expression is diminished by one-third in NOS2 −/− mice compared with nTG mice [10]. In the present study, microglial and astrocyte activation in the brain, which are associated with severe synuclein pathology, were dramatically reduced in Syn A53T /NOS2 −/− mice compared with Syn A53T mice. It is possible that brain regionspecific synuclein aggregation and pathology contribute to Iba-1/GFAP expression when nos2 is knocked out.
Another possibility is that unknown synuclein pathology/ NOS2-associated molecular targets contribute to glial hypoactivity/degradation when nos2 is deleted in vivo. Future studies will focus on identifying the molecules that contribute to glial inactivation and the amelioration of synuclein pathology when nos2 is deleted. Overall, the available data suggest that NOS2 has critical functions in the modulation of glial homeostasis in this mouse model of PD.
In conclusion, we generated Syn A53T /NOS2 −/− mice for the first time by crossing human α-synuclein A53T mutant mice and nos2 knockout mice and found that α-synuclein pathology, neuroinflammatory responses, and neuroinflammation-associated gene expression were reduced in the double transgenic mice compared with Syn A53T mice. Our data indicate that NOS2 may be a therapeutic target for modulating PD pathology in the brain. and valuable comments on our manuscript and for technical assistance with in vivo studies.

Availability of data and materials
All data generated and/or analyzed during this study are included in this published article and its supplementary information. The materials and methods are presented in Additional file 1.

Declarations Ethics approval and consent to participate
All animal experiments were approved by the Institutional Animal Care and Use Committee of the Korea Brain Research Institute (KBRI) (Approval Nos. IACUC-18-0007 and IACUC-19-0010).

Consent for publication
Not applicable.